1. Technical Field of the Invention
The present invention relates to a process for evaluating the damage induced in the skin and/or the damage caused by type A ultraviolet radiation.
2. Description of the Prior Art
Solar radiation is composed, inter alia, of type A ultraviolet radiation with wavelengths ranging from 320 nm to 400 nm (UV-A) and type B ultraviolet radiation has wavelengths ranging from 280 to 320 nm (UV-B).
U-B radiation is highly energetic and poorly penetrating. It is not represented to a significant extent in sunlight, but is dependent on climatic variations (cloudy and overcast weather and the like). The presence of UV-B radiation also varies according to the time of day (i.e., notion of peak (zenith)).
UV-A radiation is less energetic than UV-B radiation, but more penetrating and it is present in a greater amount in sunlight (at least 100 times more UV-A than UV-B). UV-A radiation is also less dependent on climatic variations and is present regardless of the time of day.
It is known that solar radiation is responsible for certain beneficial effects on the skin, such as, for example, skin darkening or tanning, but it is also capable of inducing damage in the skin, particularly in the case of so-called "sensitive" skin or of skin continuously exposed to sunlight.
In terms of beneficial effects or advantages, darkening of the skin, commonly called tanning, is an essential element in the skin's system of defense. Indeed, in response to ultraviolet radiation, the melanocytes of the top layer of the epidermis synthesize melanin which, once incorporated into the keratinocytes, act as a screening agent which is situated on the surface of the skin. The screening agent absorbs ultraviolet radiation. The purpose of the absorption is to reduce the quantity of ultraviolet radiation which crosses the skin layers in order to prevent the ultraviolet radiation from reaching the deep layers of the skin and thereby causing damage to the skin.
With respect to the harmful effects of ultraviolet radiation, it is known that excessive exposure of the skin to ultraviolet radiation, especially solar radiation, can lead to a change in the elasticity of the skin, and in the content of certain compounds within the skin, and thus promote the acceleration of the natural skin aging process. This accelerated or premature aging process due to UV radiation is generally called photoaging or actinic aging or dermatoheliosis.
Photoaging therefore results from the action of extrinsic factors on the skin, including solar radiation and, in particular, UV rays.
Photoaging is the consequence of the repetition of defined events, induced by these extrinsic factors, in particular, solar radiation, which defined events have up until now been assumed but never demonstrated.
Defined events are understood to mean any single or isolated event resulting from irradiation by UV radiation.
In man, photoaging causes the skin to have a dry, rough clinical appearance associated with a loss of elasticity, as well as marked wrinkles.
The histological signs of photoaging are (for a review, Gilchrest B. A., Skin and Aging Processes, 1989, CRC Press) at the epidermal level: the variation in the thickness of the epidermis (atrophy or hyperplasia according to the zones observed), a cellular atypia (Kligman L. M. and Kligman A. M., Photodermatol., 1986, 3: 215-227), a loss of cell polarity, an unevenness of the horny layer, a reduction in the number of Langerhans' cells (Lavker R. M. et al., J. Invest. Dermatol., 1987, 88: 44s-51s), a pigmentation characterized by a mosaic appearance with hypo- or hyperpigmentation zones, and a linearization of the dermoepidermal junction (Lavker R. M., J. Invest. Dermatol., 1979, 73: 59-).
The epidermal impairments are in fact relatively minor compared with the dermal impairments which are the most obvious and the most substantial (Kligman L. M. and Kligman A. M., Photodermatol., 1986, 3: 215-227).
It is indeed at the level of the extracellular matrix that the principal effects are observed. The fibroblasts are hyperactive (Kligman L. M. and Kligman A. M., Photodermatol., 1986, 3: 215-227). The collagen is reduced in quantity (Oikarinen A. et al., Photodermatol., 1985, 2: 15-26, Warren R. et al., J. Am. Acad. Dermatol., 1991, 25: 751-760). The solubility of the fibers is reduced (Oikarinen A. and Kallioinen M., Photodermatol., 1989, 6: 24-31), and a basophilic degeneration is observed.
The ultrastructural impairments of collagen result in a decrease in the number of fibrils, a reduction in the electron density, a reduction in the transverse striations (Mitchell R. E., J. Invest. Dermatol., 1967, 48: 203-220). Conversely, the elastic tissue undergoes hyperplasia (Kligman A. M., J.A.M.A., 1969, 210: 2377-2380, Warren R. et al., J. Am. Acad. Dermatol., 1991, 25: 751-760). The middle dermis is characterized by a regrouping of fibers, thus forming a superficial zone called "grenz zone", which fibers are absent from the papillary dermis. In the deepest part of the dermis, the elastic tissue is abnormal and disorganized (Chen V. L. et al., J. Invest. Dermatol., 1986, 87: 334-337), but in particular, it is present in a large quantity, which results histologically in a mass of elastic tissue. This phenomenon is known as actinic elastosis (Braverman I. M. and Fonferko E., J. Invest. Dermatol., 1982, 78: 434-443; Matsuoka L. Y. and Uitto J., Aging and the Skin, Balin A. K. and Kligman A. M. Eds., Raven Press N.Y. 1989, 7, 141-151). Elastin has a granular appearance (elastotic material) and inclusions in electron microscopy (Braverman I. M. and Fonferko E., J. Invest. Dermatol., 1982, 78: 434-443). The glycosaminoglycans and the proteoglycans are increased (Smith J. G. et al., J. Invest. Dermatol., 1962, 39: 347-350, Sams W. M. and Smith J. G., J. Invest. Dermatol., 1961, 37: 447-452).
The presence of a dermal infiltrate composed of mastocytes, lymphocytes and histiocytes has often been observed (Lavker R. M. and Kligman A. M., J. Invest. Dermatol., 1988, 90: 325-330). The blood vessels are also modified (Braverman I. M. and Fonferko E., J. Invest. Dermatol., 1982, 78: 444-448). In particular, their number is reduced, the endothelial cells are dilated and the wall of the vessels is thickened and a lamination of the basal membrane of the endothelial cells is observed.
However, even if the clinical and histological signs of photoaged skin have been well studied, the processes which lead to these signs remain unknown to this day. In particular, the respective role of UV-A and UV-B in these processes has not been demonstrated.
This is largely due to the difficulty in using a simple and reliable study model.
Animal models developed with the aim of reproducing the damage caused by UV radiation and with a pharmacological aim to test "antiaging" molecules are known in the prior art (Sams W. M. et al., J. Invest. Dermatol., 1964, 43: 467-471; Kligman L. H., Yearly review, the hairless mouse and photoaging; Photochem. Photobiol., 1991, 54(6), 1109-1118).
However, these models require the use of laboratory animals, which may present ethical problems.
Furthermore, to obtain a photoaged image which is close to what is real, it is necessary, with these models, to perform the irradiation chronically or continuously for several weeks, which makes the use of such models cumbersome.
The animals used in these models are generally mice, whose cells are different, in a number of respects, from human cells.
Furthermore, the skin of mice is much finer than human skin and this parameter should be considered when evaluating the effects of UV radiation, because these effects depend on the penetration of the rays through the skin.
The dermis of mice is different from the human dermis (papillary and cross-linked dermis does not exist in mice) and can have, depending on the species used in the model, numerous residues of degenerate hair follicles.
Finally, these models do not give any precise indications on the early events which lead to the histological image of photoaging.
Study models in man are also known (Scharffetter K. et al., Arch. Dermatol. Res., 1991, 283: 506-511; Korwin-Zmijowska C. et al., Nouv. Dermatol., 1993, 12: 487), but those models are difficult to use. The use of human volunteers can present ethical difficulties and unavoidably brings about constraints for the subject.
Furthermore, the variability between individuals (skin phototype, age of the subject, too much space medical history, ongoing treatments and the like) is such that the reproducibility of the results may not be achieved.
Finally, in vitro models are known. Some of these models require cell cultures in which the effects of the UV irradiation on one or more cell markers in the culture are studied.
The effects of the UV-A radiation have thus been studied on the keratinocytes (epidermal cells), the fibroblasts (cells of the dermis), Langerhans' cells (immunocompetent cells of the epidermis), or the melanocytes (pigment cells) (Gilchrest B. A., J. Gerontol., 1980, 35: 537-541; Scharffetter K. et al., Arch. Dermatol. Res., 1991, 283: 506-511; Petersen M. J. et al., J. Invest. Dermatol., 1992, 99: 440-444; Nascimento A. et al., Nucleic Acids Res., 1993, 21(5), 1103-1109).
The reason why such models cannot reflect what is really happening can be readily understood. These models are cultures of monolayer cells, which are very different from an actual tissue, especially with its three-dimensional structure. Generally, cultures are produced from a single cell population, which also does not reflect reality.
Accordingly, the influence of the thickness of the cells affected by the radiation and the role of the penetration of UV radiation into the skin, which is obviously dependent on the thickness of the cell layers which must be penetrated cannot be studied using such models.
The absence of a matrix context, particularly with respect to the dermal fibroblasts which are essential for their metabolic activity, distorts the interpretations of the results obtained; it is indeed difficult to extrapolate the data obtained on plastic in vitro for a real situation in vivo in man.
It is impossible, with this type of model, to conduct product applications or studies by the topical route of administration.
Finally, some of these models require cell lines, that is to say genetically modified cells which also cannot reflect the physiological reality of normal cells.
Models using epidermis or skin equivalents are also known. With respect to the models using epidermis equivalents, their principal disadvantage results from the fact that they do not represent what is real because of the absence of a dermis or of dermis equivalent (Noel-Hudson M. S. et al., Nouv. Dermatol., 1993, 12: 493). The tanning tests developed on epidermis equivalents containing melanocytes (FR 2 689 904) may also be noted.
Other studies use skin equivalents (Mammone, et al., J. Invest. Dermatol., 1992, 98(4), 655; Ridge J. M. et al., Clin. Res., 1992, 40(2): 543A; Reece B. et al., J. Soc. Cosmet. Chem., 1992, 43: 307-312; Pelle E. et al., J. Invest. Dermatol., 1993, 100, (4), 595; Nelson D. et al., Photochem. Photobiol., 1993, 57(5): 830-837); Haake and Polakowska, Cell death and differentiation, 1995, vol. 2, 183-193).
Independently of the model selected, which is sometimes quite different from normal skin, these studies have most often been very brief and have never studied the specific influence of UV-A radiation on markers linked to photoaging.
Accordingly, before the date of the present invention, no study model has made it possible to study and understand the events whose repetition leads to clinical and histological signs of photoaging.
The human skin consists of two compartments, namely a superficial compartment, the epidermis, and a deep compartment, the dermis.
The natural human epidermis is mainly composed of three types of cells which are the keratinocytes, which are highly predominant, the melanocytes and the Langerhans' cells. Each of these cell types contributes, through its specific functions, to the essential role played in the body by the skin.
The dermis provides a solid support to the epidermis. The dermis also its nutrient-providing component. It consists mainly of fibroblasts and an extracellular matrix itself composed mainly of collagen, elastin and of a substance (called ground substance), which components are synthesized by their fibroblasts. Leukocytes, mastocytes or tissue macrophages also exist therein. It also consists of blood vessels and nerve fibers. In normal skin, that is to say, skin which is neither pathologic nor cicatricial, the fibroblast is in a quiescent state, that is to say is nonproliferative, is not very active from a metabolic point of view and is not mobile.
The epidermis is the first target reached by solar radiation, particularly, UV radiation.
The weakly penetrating UV-B radiation reaches mainly the epidermis. The role of the UV-B radiation has been clearly demonstrated in the induction of UV-induced skin cancers. It has, in fact, as a principal chromophore, nucleic acids, in particular, deoxyribonucleic acid, in which it induces lesions and/or mutations (Eller M. S., 1995, in Photodamage., 26-56, Blackwell ed.)
In contrast, the role of the UV-A radiation in the induction of the defined events whose repetition leads to the photoaging phenotype is not known, even if its high penetrating power suggests that it reaches the dermis in which it induces its damaging effects.